1. Field of the invention:
The present invention relates to a thermostable DNA polymerase and a method for producing the same. More particularly, the present invention relates to an excellent thermostable DNA polymerase which is produced by microorganisms of the genus Thermus, for example, Thermus thermophilus, and a method for the production of this DNA polymerase.
The thermostable DNA polymerase of the present invention is an enzyme which catalyzes a synthesis of DNA complementary to a template DNA from the template DNA primer and four varieties of deoxyribonucleoside triphosphates. This enzyme is used in gene manipulation techniques such as the amplification of nucleic acid sequences and the determination of DNA base sequences.
2. Description of the prior art:
Thermostable DNA polymerase derived from Thermus aquaticus YT-I (ATCC 25104) has been known previously. A. Chien et al. (J. Bacteriol. Vol. 127, pp. 1550-1557, 1976) reported a method for the isolation and purification of DNA polymerase from Thermus aquaticus YT-I, and also reported on the enzymatic properties (i.e., optimal temperature optimal pH and effects of divalent ion concentrations, etc.) of this polymerase.
Nucleic acid sequences can be effectively amplified by using such thermostable DNA polymerases. A method of amplifying nucleic acid sequences using the above-mentioned thermostable DNA polymerase derived from Thermus aquaticus YT-I (hereinafter referred to as Taq polymerase) has been reported by Saiki et al. (Science, Vol. 230, pp. 1350-1354, 1985; Bio/Technology, Vol. 3, pp. 1008-1012, 1985; and Japanese Laid-Open Patent Publication No. 63-102677). This method includes the following processes.
1) A double-stranded DNA sequence is denatured to prepare a single-stranded sequence. PA0 2) The single-stranded template nucleotide chain so obtained is annealed with a primer. PA0 3) The said primer is elongated.
In this procedure, heat is applied during process 1, while DNA polymerase acts in process 3. Since the said DNA polymerase is thermostable, deactivation hardly occur in returning to process 1 subsequent to process 3. That is, the successive processes 1 through 3 can be repeated continuously, and therefore the amplification of the nucleotide chain can be effectively performed. However, even if Taq polymerase is used when the processes 1 through 3 are continuously repeated, particularly in the denaturation process 1 where temperatures of about 90.degree. to 105.degree. C. are applied, enzymatic activity does diminish and therefore adequate amplification cannot be obtained.
Thus, an adequately thermostable DNA polymerase has not previously been obtained, and the development of a DNA polymerase of high thermostability is desirable.